Transforming Perchlorate Reducing Pathway into the E.coli Chassis

CPB-38-441 Expression Vector

Our cloning strategy was to replace the “Tinsel” chromophore gene cassette contained in the host plasmid CPB-38-441,with the G-Blocks for Cld(SP+) and Cld(SP-) using the compatible BsaI sticky-ends located at the ends of our G-Blocks and the Tinsel gene cassette as shown in Figure_ below. When transformed and plated, we expected that ligation mixtures containing equal molar concentrations of digested G-Block and plasmid would produce an equal number of white colonies (Cld-containing plasmids) and purple colonies (Tinsel containing plasmids)in the presence of kanamycin. We also plated transformations that contained either no DNA or the host plasmid CPB-38-441. The absence of colonies on the former would verify that resulting colonies are not due to contamination. The presence of purple colonies on the latter would demonstrate that the lack of colonies on either Cld plate was not due to failed transformation but rather a problem upstream in the cloning procedure.

CLD± Constructs

Figure_. Shown above are the expected fragments resulting from the BsaI digestion of the Cld G-Blocks and the CPB-38-441 plasmid. Both G-Blocks and plasmid have compatible stick-ends that assure their replacement with the Tinsel cassette in the correct orientation and at single copy.

Plating

The plating results are shown below (Figure_). As expected white and purple colonies were present for both Cld(SP+) and Cld(SP-) ligations. The observation that no colonies were detected on the “No DNA control showed that these colonies were not contaminants but derived from the DNA that we added intentionally. The presence of purple colonies on the positive control plate showed that the transformation procedure was functional. We observed however, that on our recombinant DNA plates, there were fewer white colonies than purple colonies. Our supervisor suggested that this may be because of partially synthesized G-Block DNA that is missing the terminal BsaI sites.

Figure_. The schematic on the left display the two possible ligation products (gene replacement with Cld and Tinsel re-ligation). Images on the right show kanamycin LB agar plates for the “No DNA” negative control (-)ctrl, the host plasmid control [(+)ctrl], the Cld(-SP) ligation and the Cld(+SP) (bottom right).

	Vial Number	260/280	Concentration in nanograms/microlitre
Read 1	1	1.78	86.7
Read 2	1	1.83	85.4
Read 1	2	1.79	98.5
Read 2	2	1.79	98.6
Read 1	3	1.83	81.9
Read 2	3	N/A	N/A
Read 3	3	1.82	82.9
Read 1	4	1.82	97.1
Read 2	4	1.80	97.3
Read 1	5	1.82	92.7
Read 2	5	1.82	93.9
Read 1	6	1.82	95.4
Read 2	6	1.82	95.4

Gel Analysis

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